Riboswitches are a class of non-protein-coding (nc) RNA elements involved in the regulation of key metabolic pathways in bacteria through small molecule binding. Several of these pathways, such as preQ1 biosynthesis, are unique to eubacteria and represent novel antimicrobial targets to combat human pathogens. To better understand this unique mechanism of gene regulation, we determined ligand-bound and ligand-free crystal structures of a class 1 preQ1 riboswitch during the prior 2-year ARRA funding period. This work provided the first analysis of a preQ1 riboswitch that controls translation, and represents the only structural analysis of a translational riboswitch in the ligand-free (apo) stat. The results provide a tantalizing glimpse of how a cellular metabolite can govern the interaction between mRNA and the ribosome. A comparison of the ligand-bound and ligand-free structures suggested that the conformation of the preQ1-binding pocket is dictated by ligand binding, and that this interaction controls access to a spatially distant ribosome-binding site (RBS). In Aim #1 of this proposal, we will expand on prior work to evaluate whether a phylogenetically unrelated preQ1 class 2 riboswitch employs the RBS-sequestration mechanism, thereby testing the universality of this gene regulation strategy. In Aim #2, we will examine the interaction between preQ1 riboswitches and the translation-initiation complex. The recognition of mRNA by the ribosome is a fundamental process, yet no investigation has probed the influence of ribosomes on the riboswitch folding landscape. We have assembled a team of single molecule (sm)FRET and ribosome experts for this aim (Nils Walter, University of Michigan and Dmitri Ermolenko, University of Rochester), and the results should provide a meaningful description of the ligand levels and riboswitch sequences required for translational regulation. Aim #3 of this proposal is based on our prior analysis of the apo preQ1 riboswitch. Obtaining the apo crystal structure was unexpected since independent studies on transcriptional preQ1 riboswitches showed strict, ligand-dependent folding. For this reason, we used small angle X-ray scattering to demonstrate that the translational preQ1 riboswitch is as compact in solution as the ligand-bound state. However, the apo crystal structure could not adequately recapitulate the apo riboswitch scattering data, suggesting the ligand-free ensemble in solution is complex. In Aim #3, we will analyze the solution ensembles of preQ1 riboswitches by small angle scattering and in-line probing. This work will require novel computational approaches that will be conducted in collaboration with David Mathews (University of Rochester). These experiments will provide insight into the 'prefoldedness' of the apo state and its receptiveness to ligand binding, as embodied by the principle of conformational selection. The overall results of the proposed investigation will expand our understanding of this unique mechanism of gene regulation for a novel metabolic pathway in bacteria. In the long term, this work can be exploited for the development of novel antimicrobials that have little or no effect on host pathways.